Interaction studies between human papillomavirus virus-like particles and laminin 332 by affinity capillary electrophoresis assisted by bio-layer interferometry.

Human papillomavirus (HPV) interacts, in vitro, with laminin 332 (LN332), a key component of the extracellular matrix. In this study, we performed bio-layer interferometry (BLI) and affinity capillary electrophoresis (ACE) to investigate the binding properties of this interaction. Virus-like particles (VLPs), composed of the HPV16 L1 major capsid protein, were used as HPV model and LN332 as the VLPs binding partner. Using BLI, we quantitatively determined the kinetics of the interaction, via the measurement of VLP binding and release from LN332 immobilized onto the surface of aminopropylsilane biosensors. We found an averaged kon of 1.74 x 104 M-1s-1 and an averaged koff of 1.50 x 10-4 s-1. Furthermore, an ACE method was developed to study the interaction under physiological conditions, where the interactants are moving freely in solution, without any fluorescence labeling. Specifically, a constant amount of HPV16-VLPs was preincubated with increasing LN332 concentrations and then the samples were injected in the capillary electrophoresis instrument. A shift in the migration time of the HPV16-VLP/LN332 complexes, carrying an increasing number of LN332 molecules bound per VLP, was observed. The mobility of the complexes was found to decrease with increasing LN332 concentrations in the sample. It was used to quantify stability constant. From BLI and ACE approaches, we reported an apparent equilibrium dissociation constant in the nanomolar range (8.89 nM and 17.7 nM, respectively) for the complex between HPV16-VLPs and LN332.

Development of 3D printed mini-waffle shapes containing hydrocortisone for children's personalized medicine.

Hydrocortisone is mainly used in the substitution treatment of adrenal insufficiency which results in a dysregulation of cortisol. Compounding of hydrocortisone capsules remains the only low-dose oral treatment suitable for the pediatric population. However, capsules often show non-compliance in mass and content uniformity. Three-dimensional printing offers the prospect of practising personalized medicine for vulnerable patients like children. The goal of this work is to develop low-dose solid oral forms containing hydrocortisone by hot-melt extrusion coupled with fused deposition modeling for the pediatric population. Formulation, design and processes temperatures were optimized to produce printed forms with the desired characteristics. Red mini-waffle shapes containing drug loads of 2, 5 and 8 mg were successfully printed. This new 3D design allow to release more than 80 % of the drug in 45 min indicating a conventional release like the one obtained with capsules. Mass and content uniformity, hardness and friability tests complied with European Pharmacopeia specifications, despite the considerable challenge of the small dimensions of the forms. This study demonstrates that FDM can be used to produce innovative pediatric-friendly printed shapes of an advanced pharmaceutical quality to practice personalize medicine.

Validation of an LC-MS/MS Method Using Solid-Phase Extraction for the Quantification of 1-84 Parathyroid Hormone: Toward a Candidate Reference Measurement Procedure.

BACKGROUND: Parathyroid hormone (PTH) measurement is important for patients with disorders of calcium metabolism, including those needing bone-turnover monitoring due to chronic kidney disease-mineral bone disorder. There are currently 2 generations of PTH immunoassays on the market, both having cross-reactivity issues and lacking standardization. Therefore, we developed an LC-MS/MS higher-order method for PTH analysis. METHODS: The method was calibrated against the international standard for 1-84 PTH (WHO 95/646). Antibody-free sample preparation with the addition of an isotope-labeled internal standard was performed by solid-phase extraction. Extracts were analyzed by LC-MS/MS. EDTA-K2 plasma was used throughout the development and validation. Bias and uncertainty sources were tested according to ISO 15193. Clinical Laboratory Standards Institute guidelines and reference measurement procedures were consulted for the design of the validation. Patient samples and external quality controls were compared between LC-MS/MS and 2 third-generation immunoassays. RESULTS: The method was validated for 1-84 PTH from 5.7 to 872.6 pg/mL. The interassay imprecision was between 1.2% and 3.9%, and the accuracy ranged from 96.2% to 103.2%. The measurement uncertainty was <5.6%. The comparison between LC-MS/MS and the immunoassays showed a proportional bias but moderate to substantial correlation between methods. CONCLUSIONS: This LC-MS/MS method, which is independent of antibodies, is suitable for a wide range of PTH concentrations. The obtained analytical performance specifications demonstrate that development of a reference measurement procedure will be possible once a higher order reference standard is available.

Qualitative and quantitative comparison of different commercially available 1-84 parathyroid hormone proteins to the WHO international standard 95/646 using orthogonal methods.

Relative quantitation methods rely on the use of reference substances to determine the content of samples. The aim of this study was to compare 1-84 parathyroid hormone (PTH) standards from different manufacturers to the WHO international standard 95/646. CE and LC with UV detection were investigated as quick and inexpensive quantitation methods, with an emphasis on selectivity between intact 1-84 PTH and its oxidized forms. Both methods were fully validated according to ICH Q2R1. Moreover, method performance was also evaluated according to guidelines defining the maximum allowable measurement uncertainty (MU) of a biological parameter from its intraindividual variation (CVI), as well as the proportion of that MU devoted to the reference material. This study highlighted the fact that some 1-84 PTH standards have a content that is actually twice as high as the one stated on the label, which was confirmed by an amino acid analysis investigation. Our approach offers a quick and inexpensive way to estimate the content of 1-84 PTH standards.

Improvement of chemo- and stereoselectivity for phosphorothioate oligonucleotides in capillary electrophoresis by addition of cyclodextrins.

Phosphorothioate (PS) modification is one of the most widely used oligonucleotide chemical alterations in the oligonucleotide backbone. It has proven to be crucial in the field of therapeutic oligonucleotides regarding the optimization of their physicochemical and biological properties. In this study, a capillary electrophoresis (CE) method with an acidic background electrolyte (BGE) containing a combination of ß- and γ-cyclodextrins derivatives as chiral selectors is proposed for the diastereomeric separation of 5-mer oligonucleotides containing 0, 1, 2, or 3 phosphorothioate linkages (5´-TCGTG-3´). The effects of the BGE pH, organic modifier addition, and type of cyclodextrin (CD) on chemo- and stereoselectivity and resolution were studied. A mixture of 25 mM (2-hydroxy-3-N,N,N-trimethylamino)propyl-γ-CD and 10 mM carboxymethyl-ß-cyclodextrin in a pH 3 buffer was found to be the most appropriate system for the qualitative evaluation of the short oligonucleotides investigated. These phosphorothioate oligonucleotides were separated with high efficiency in less than 11 min with no capillary treatment. The suggested approach can be the basis for purity testing of this new generation of therapeutics.

Comparison of Three Complementary Analytical Techniques for the Evaluation of the Biosimilar Comparability of a Monoclonal Antibody and an Fc-Fusion Protein.

In this work, a monoclonal antibody, adalimumab, and an Fc-fusion protein, etanercept, were studied and compared to one of their biosimilars. Samples submitted to stress conditions (agitation and high temperature) were used for method development. The developed methods were also applied to samples reduced by beta-mercaptoethanol to evaluate their capability to distinguish the expected species. Capillary gel electrophoresis (CGE), reversed-phase liquid chromatography (RPLC), and size-exclusion chromatography (SEC) methods coupled with UV detection were used to analyze the biopharmaceuticals. Their complementarity was investigated. For further molecular weight determination, SEC-multi angle light scattering and RPLC-quadrupole time-of-flight were occasionally used. For adalimumab, a larger amount of fragments and aggregates was observed in the biosimilar compared with the reference product. For etanercept, more related species were found in the reference product. Those three separation techniques showed good complementarity. Indeed, RPLC enabled the separation of hydrophilic and hydrophobic degradation products. CGE provided good selectivity for several adalimumab fragments, and SEC was useful for the analysis of aggregates and certain fragments that cannot be separated by the other approaches. Moreover, those formulations were submitted to mild stress conditions (30°C, 300 rpm for 4 h) that mimic shipping conditions. No additional peak was found under these conditions for the two studied biopharmaceuticals.

Blood Microsampling to Monitor Metabolic Profiles During Physical Exercise.

Monitoring approaches and technical improvements are key factors to improve a sportsman's health, training, and recovery after an injury. In this study, a targeted metabolomics approach using microsampling with hemaPEN® was developed to measure changes in blood concentrations of nine amino acids and four organic acids before, during, and after exercise. The aim of this research project was to investigate if a reliable monitoring of metabolite levels during sports activity can be achieved by collecting one drop of whole blood at different time points. A hemaPEN device is an easy-to-use and noninvasive microsampling technique designed to collect four accurate and precise blood volumes simultaneously (10.96 µl). Twenty healthy volunteers between 19 and 30 years of age were included in this study. Physical activity consisted in running as fast as possible 1,600 m after 400 m warm-up. One drop of blood was collected at five time points: before exercise, after 800-m running, after 1,600 m, and 30 min and 60 min after finishing the exercise. The influence of physical activity on metabolite levels was evaluated using two ultrahigh-performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) methods. Analytical performance criteria such as metabolite stability, method precision, trueness, and accuracy were found to be satisfactory. Expected significant metabolic changes were identified for lactic acid, main TCA cycle intermediates, and some amino acids (e.g., creatinine, choline, and taurine). This preliminary study performed on a small cohort demonstrated a high interest of using microsampling for fluxomics analysis, not only to collect quickly and easily biological samples during sports events but also because it is much easier to store and to process the samples than classical plasma/serum samples obtained by venipuncture. The present results open new avenue for fluxomics analysis in the context of health care.

Analytical techniques currently used in the pharmaceutical industry for the quality control of RNA-based therapeutics and ongoing developments.

The number of RNA-based therapeutics has significantly grown in number on the market over the last 20 years. This number is expected to further increase in the coming years as many RNA therapeutics are being tested in late clinical trials stages. The first part of this paper considers the mechanism of action, the synthesis and the potential impurities resulting from synthesis as well as the strategies used to increase RNA-based therapeutics efficacy. In the second part of this review, the tests that are usually performed in the pharmaceutical industry for the quality testing of antisense oligonucleotides (ASOs), small-interfering RNAs (siRNAs) and messenger RNAs (mRNAs) will be described. In the last part, the remaining challenges and the ongoing developments to meet them are discussed.

Development of a sensitive MEKC-LIF method for synthetic cathinones analysis.

Synthetic cathinones are phenylalkylamine compounds related to natural cathinone from Catha edulis leaves. Due to their sympathomimetic effects comparable to common illicit drugs, these substances are mainly drugs of abuse and constitute the second most frequently seized group of new psychoactive substances. In order to ensure their regulation and to promote public health, reliable analytical tools are required to track these substances. In the present study, we developed a CE hyphenated to laser-induced fluorescence detection method to demonstrate its suitability to perform fast and cost-effective synthetic cathinones analysis. Fourteen compounds including isobaric compounds and position isomers were selected to encompass the large panel of chemical structures. To separate the FITC-labeled analytes (presenting the same negative charge and close mass to charge ratios), MEKC separation mode was selected. Method selectivity was not suitable using common surfactants. In this context, alkyl polyethylene glycol ether surfactants were successfully used as neutral surfactant to overcome this analytical challenge. The effect of surfactant nature on separation performances and migration behaviors of the analytes was also studied. Optimal BGE composition included 75 mM borate buffer at pH 9.3 and 0.4 mM of C12E10 surfactant. Final MEKC separation conditions were proposed to analyze a large panel of synthetic cathinones. This method helped to reach a sensitivity with LOD from 0.1 to 0.4 nM (pg/mL order).

Capillary Electrophoresis-Mass Spectrometry at Trial by Metabo-Ring: Effective Electrophoretic Mobility for Reproducible and Robust Compound Annotation.

Capillary zone electrophoresis-mass spectrometry (CE-MS) is a mature analytical tool for the efficient profiling of (highly) polar and ionizable compounds. However, the use of CE-MS in comparison to other separation techniques remains underrepresented in metabolomics, as this analytical approach is still perceived as technically challenging and less reproducible, notably for migration time. The latter is key for a reliable comparison of metabolic profiles and for unknown biomarker identification that is complementary to high resolution MS/MS. In this work, we present the results of a Metabo-ring trial involving 16 CE-MS platforms among 13 different laboratories spanning two continents. The goal was to assess the reproducibility and identification capability of CE-MS by employing effective electrophoretic mobility (µeff) as the key parameter in comparison to the relative migration time (RMT) approach. For this purpose, a representative cationic metabolite mixture in water, pretreated human plasma, and urine samples spiked with the same metabolite mixture were used and distributed for analysis by all laboratories. The µeff was determined for all metabolites spiked into each sample. The background electrolyte (BGE) was prepared and employed by each participating lab following the same protocol. All other parameters (capillary, interface, injection volume, voltage ramp, temperature, capillary conditioning, and rinsing procedure, etc.) were left to the discretion of the contributing laboratories. The results revealed that the reproducibility of the µeff for 20 out of the 21 model compounds was below 3.1% vs 10.9% for RMT, regardless of the huge heterogeneity in experimental conditions and platforms across the 13 laboratories. Overall, this Metabo-ring trial demonstrated that CE-MS is a viable and reproducible approach for metabolomics.

Enhancing protein discoverability by data independent acquisition assisted by ion mobility mass spectrometry.

Ion mobility (IM) mass spectrometry allows conducting data independent acquisition (DIA) where all ions entering the instrument are fragmented based on their drift time. In this work, DIA operational parameters were first optimized using a design of experiments. The optimization of data treatment involved a smoothing algorithm of the IM dimension, which increased the number of identified peptides. Then, classical DDA and IM-based DIA were compared injecting increasing amounts of a complex proteome digest (E. coli). Results revealed that compared to DDA, DIA allowed to identify from 2 to 3.3 times more proteins, depending on the injected quantity. To evaluate proteome coverage, endogenous proteins in E. coli cells were sorted by abundance deciles. A large majority of the proteins uniquely observed in DDA were part of the 10% most abundant protein groups. Interestingly, owing to the absence of ion-picking algorithm, DIA allowed to identify proteins coming from a broader concentration range therefore greatly improving proteome coverage. Furthermore, ion mobility separation improved coverage by separating co-eluting peptides. Physicochemical properties of peptides uniquely detected by DIA or DDA were also compared using supervised and unsupervised multivariate analysis. As a result, peptides having a higher mass and being relatively hydrophobic were significantly more identified in DIA. Finally, semi-quantitative performance of both methods was investigated and proved to be comparable, except that DIA demonstrated a better sensitivity than DDA. As a conclusion, we demonstrated in this study that both acquisition modes provide complementary information about the proteome under investigation.

Hyphenation of capillary zone electrophoresis with mass spectrometry for proteomic analysis: Optimization and comparison of two coupling interfaces.

Capillary electrophoresis tandem mass spectrometry (CE-MS/MS) is an interesting tool for proteomic analysis as the separation principle is orthogonal to liquid chromatography tandem mass spectrometry (LC-MS/MS). The combination of both techniques can bring complementary information to enlarge proteome coverage. In this study, sample preconcentration techniques were investigated in order to improve sample loading and therefore sensitivity. Dynamic pH junction (DPJ) was found to be the most interesting approach by using 200 mM ammonium acetate (NH4Ac) adjusted to pH 10.0 as sample matrix. The use of DPJ allowed the identification of more peptides and proteins compared to conventional injections. Moreover, the sheath liquid (SL) composition was optimized in order to enhance signal intensity. A nanoflow SL interface (EMASS-II) was compared to the traditional coaxial SL interface (Triple tube) in terms of number of identified and proteins as well as detection sensitivity (peak area and peak height). MS acquisition was performed using both data-dependent acquisition (DDA) and data-independent acquisition (DIA) modes. The results showed that the combined use of these two acquisition modes provided additional information in terms of identification. Moreover, the use of EMASS-II interface allowed the identification of approximately two times more peptides and proteins. Besides, there was an improvement in sensitivity using EMASS-II as peak height and peak area were improved by 4 and 6-fold, respectively, compared to the Triple tube. Altogether, by combining an efficient sample preconcentration method, a nanoflow CE-MS interface and a hybrid ion-mobility qTOF mass spectrometer, a satisfying sequence coverage was obtained by analyzing 1 µg of E. coli proteome digest.

Evaluation of hydrophilic interaction liquid chromatography, capillary zone electrophoresis and drift tube ion-mobility quadrupole time of flight mass spectrometry for the characterization of phosphodiester and phosphorothioate oligonucleotides.

Oligonucleotide-based medicines that can modulate gene expression have numerous potential applications in targeted therapies. Most of the commercialized therapeutic oligonucleotides are chemically modified to increase their in vivo lifetime. In this work, we studied poly-deoxy(thymidylic) acids (dT) and modified phosphorothioate oligonucleotides (PS). Several analytical techniques, including ion-pair reverse phase liquid chromatography, are described in the literature to assess their quality but most of them present significant drawbacks. In the present study, dT and PS mixtures were analyzed by hydrophilic interaction liquid chromatography (HILIC) and capillary zone electrophoresis (CZE) coupled to ultraviolet detection. In HILIC, the selectivities of three types of stationary phases (dihydroxypropane, phosphorylcholine and amide) were compared. Optimal conditions were determined and consisted of an amide stationary phase with a mobile phase made up of water, acetonitrile and 15 mM ammonium acetate (pH 5.5). In those conditions, high resolving power and good repeatability were achieved. In CZE, the effect of the background electrolyte (BGE), its pH and concentration were evaluated. A BGE made up of 300 mM ammonium acetate adjusted to pH 6.0 was selected. Finally, the two techniques were compared in terms of selectivity, repeatability and peak efficiency. In the second part of the study, HILIC and CZE were both coupled to a drift-tube ion-mobility quadrupole time-of-flight MS detector (DTIMS-QTOF) to assess the added value of this coupling for oligonucleotide characterization. Indeed, by using the measured collision cross section (CCS), the evaluation of the number of nucleotides was performed. Looking across the results, HILIC and CZE coupled to DTIMS-QTOF can be considered as promising tools for the quality control of oligonucleotides.

Production and characterization of virus-like particles of grapevine fanleaf virus presenting L2 epitope of human papillomavirus minor capsid protein.

BACKGROUND: Virus-like particle (VLP) platform represents a promising approach for the generation of efficient and immunogenic subunit vaccines. Here, the feasibility of using grapevine fanleaf virus (GFLV) VLPs as a new carrier for the presentation of human papillomavirus (HPV) L2 epitope was studied. To achieve this goal, a model of the HPV L2 epitope secondary structure was predicted and its insertion within 5 external loops in the GFLV capsid protein (CP) was evaluated. RESULTS: The epitope sequence was genetically inserted in the αB-αB" domain C of the GFLV CP, which was then over-expressed in Pichia pastoris and Escherichia coli. The highest expression yield was obtained in E. coli. Using this system, VLP formation requires a denaturation-refolding step, whereas VLPs with lower production yield were directly formed using P. pastoris, as confirmed by electron microscopy and immunostaining electron microscopy. Since the GFLV L2 VLPs were found to interact with the HPV L2 antibody under native conditions in capillary electrophoresis and in ELISA, it can be assumed that the inserted epitope is located at the VLP surface with its proper ternary structure. CONCLUSIONS: The results demonstrate that GFLV VLPs constitute a potential scaffold for surface display of the epitope of interest.

Determination of iohexol by capillary blood microsampling and UHPLC-MS/MS.

One of the most important tools used to evaluate kidney function in the context of chronic kidney disease or other renal function related pathologies is the exploration of glomerular filtration rate (GFR). Iohexol is up to this moment a good candidate molecule for the GFR assessment since it exhibits minimum protein binding rates and minimum extra-renal clearance, being neither secreted nor reabsorbed at the tubular level. This study proposes and evaluates a new LC-MS/MS method for the iohexol determination from capillary blood, prelevated using volumetric absorbative microsampling (VAMS) systems. As an alternative to VAMS, a brand new HemaPEN® device for micro-prelevation was also tested. A new high throughput sample preparation protocol adapted for iohexol quantification from whole blood VAMS samples was developed. The medium term stability study of iohexol in dried whole blood VAMS samples that was conducted showed a good stability of this molecule for up to 12 days. By collecting only 10 µL of blood, iohexol can be analyzed from dried whole blood VAMS samples for concentration ranges between 1 and 250 µg/mL. Due to the analyte stability in VAMS for up to 12 days, this approach might be successfully applied for GFR assessment for clinical cases allowing minimum invasiveness and even delayed analysis.

Targeted proteomics reveals serum amyloid A variants and alarmins S100A8-S100A9 as key plasma biomarkers of rheumatoid arthritis.

Serum amyloid A (SAA) and S100 (S100A8, S100A9 and S100A12) proteins were previously identified as biomarkers of interest for rheumatoid arthritis (RA). Among SAA family, two closely related isoforms (SAA-1 and SAA-2) are linked to the acute-phase of inflammation. They respectively exist under the form of three (α, ß, and γ) and two (α and ß) allelic variants. We developed a single run quantitative method for these protein variants and investigated their clinical relevance in the context of RA. The method was developed and validated according to regulations before being applied on plasma coming from RA patients (n = 46), other related inflammatory pathologies (n = 116) and controls (n = 62). Depending on the activity score of RA, SAA1 isoforms (mainly of SAA1α and SAA1ß subtypes) were found to be differentially present in plasma revealing their dual role during the development of RA. In addition, the weight of SAA1α in the total SAA response varied from 32 to 80% depending on the pathology studied. A negative correlation between SAA1α and SAA1ß was also highlighted for RA early-onset (r = -0.41). SAA2 and S100A8/S100A9 proteins were significantly overexpressed compared to control samples regardless of RA stage. The pathophysiological relevance of these quantitative and qualitative characteristics of the SAA response remains unknown. However, the significant negative correlation observed between SAA1α and SAA1ß levels in RA early-onset suggests the existence of still unknown regulatory mechanisms in these diseases.

Application of Dual-Cyclodextrin Systems in Capillary Electrophoresis Enantioseparations.

The enantioseparation of acidic and neutral compounds can be successfully achieved in capillary electrophoresis (CE) using dual-cyclodextrin (CD) systems. This chapter describes how to separate the enantiomers of acidic or neutral substances using dual-CD systems made up of a negatively charged CD derivative, i.e., sulfobutyl-ß-CD or carboxymethyl-ß-CD, in combination with a neutral one, namely heptakis(2,3,6-tri-O-methyl)-ß-CD. An acidic compound (carprofen) and a weakly acidic drug (pentobarbital) were selected as model compounds.

Enantioseparations in Nonaqueous Capillary Electrophoresis Using Charged Cyclodextrins.

The enantioseparation of acidic and basic compounds can be successfully achieved in nonaqueous capillary electrophoresis (NACE) using single-isomer charged ß-cyclodextrin (ß-CD) derivatives of opposite charge to that of the analytes. This chapter describes how to separate the enantiomers of three basic substances selected as model compounds, i.e., alprenolol, bupranolol, and terbutaline, using the negatively charged heptakis(2,3-di-O-acetyl-6-O-sulfo)-ß-CD (HDAS-ß-CD). The enantiomers of three acidic drugs (tiaprofenic acid, suprofen, and flurbiprofen) are resolved using a monosubstituted amino ß-CD derivative, namely 6-monodeoxy-6-mono(3-hydroxy)propylamino-ß-CD (PA-ß-CD).

Highly sensitive and selective separation of intact parathyroid hormone and variants by sheathless CE-ESI-MS/MS.

Parathyroid hormone (PTH) is a common clinical marker whose quantification relies on immunoassays, giving variable results as batch, brand, or target epitope changes. Sheathless CE-ESI-MS, combining CE resolution power and low-flow ESI sensitivity, was applied to the analysis of PTH in its native conformation in the presence of related forms. Fused silica and neutral-coated capillaries were investigated, as well as preconcentration methods such as transient isotachophoresis, field-amplified sample injection (FASI), and electrokinetic supercharging (EKS). The method for the separation of PTH and its variants was first developed using fused-silica capillary with UV detection. An acidic BGE was used to separate 1-84 PTH (full length), 7-84 PTH, and 1-34 PTH. Acetonitrile was added to the BGE to reduce peptide adsorption onto the capillary wall and transient isotachophoresis was used as analyte preconcentration method. The method was then transferred to a sheathless CE-ESI-MS instrument. When using a fused silica capillary, CE-MS was limited to µg/mL levels. The use of a neutral coating combined with FASI or EKS allowed a significant increase in sensitivity. Under these conditions, 1-84 PTH, 7-84 PTH, and 1-34 PTH were detected at concentrations in the low ng/mL (FASI) or pg/mL (EKS) range.

Selectivity evaluation of phenyl based stationary phases for the analysis of amino acid diastereomers by liquid chromatography coupled with mass spectrometry.

D-amino acids (AA) analysis is becoming more and more relevant for metabolomics, therefore new analytical tools need to be developed. A common approach to achieve AA enantioseparation is chiral derivatization. Among the chiral derivatization reagents, (+) or (-)-1-(9-fluorenyl) ethyl chloroformate ((+) or (-)-FLEC) has proved to be one of the most versatile. Suitable chiral selectivity for FLEC derivatives of amino acids could be obtained in reversed-phase HPLC using nonpolar stationary phases (C4, C8 and C18) and tetrahydrofuran (THF) based mobile phases. This study is meant to provide alternatives to the use of THF as organic modifier by evaluating the selectivity obtained on two phenyl based stationary phases for 19 FLEC-DL-AA pairs of diastereomers using UHPLC-MS. Several mobile phases consisting of ammonium acetate and different common organic solvents (acetonitrile (ACN), methanol (MeOH), 2-propanol (IPA)) were tested using gradient elution. Experimental design was employed for the optimization of the separation conditions. In the optimized conditions, complete chiral separation can be achieved for 18 out of 19 FLEC-DL-AAs in less than 30 min.